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This report describes statistical validation methods implemented on assay data for inhibition of subcellular redistribution of nuclear factor kappaB (NF kappaB) in HeLa cells. We quantified cellular inhibition of cytoplasmic-nuclear translocation of NF kappaB in response to a range of concentrations of interleukin-1 (IL-1) receptor antagonist in the presence of IL-1alpha using eight replicate rows in each four 96-well plates scanned five times on each of 2 days. Translocation was measured as the fractional localized intensity of the nucleus (FLIN), an implementation of our more general fractional localized intensity of the compartments (FLIC), which analyzes whole compartments in the context of the entire cell. The NF kappaB antagonist assay (inhibition of IL-1- induced NF kappaB translocation) data were collected on a Q3DM (San Diego, CA) EIDAQtrade mark 100 high throughput microscopy system. [In 2003, Q3DM was purchased by Beckman Coulter Inc. (Fullerton, CA), which released the IC 100 successor to the EIDAQ 100.] The generalized FLIC method is described along with two-point (minimum-maximum) and multiple point titration statistical methods. As a ratio of compartment intensities that tend to change proportionally, FLIN was resistant to photobleaching errors. Two-point minimum-maximum statistical analyses yielded the following: a Z' of 0.174 with the data as n = 320 independent well samples; Z' by row data in a range of 0.393-0.933, with a mean of 0.766; by-plate Z' data of 0.310, 0.443, 0.545, and 0.794; and by-plate means of columns Z' data of 0.879, 0.927, 0.945, and 0.963. The mean 50% inhibitory concentration (IC50) for IL-1 receptor antagonist over all experiments was 213 ng/ml. The combined IC50 coefficients of variation (CVs) were 0.74%, 0.85%, 2.09%, and 2.52% for the four plates. Repeatability IC50 CVs were as follows: day to day 3.0%, row to row 8.0%, plate to plate 2.8%, and day to day 0.6%. The number of cells required for statistically resolvable differences in dose concentrations, plotted in a family of FLIN sigma/deltamicro (SD/range) curves and tabulated, demonstrated cell-by-cell assay precision with our combined sigma/deltamicro = 0.32 that required approximately 10-fold fewer cells than in a previously reported NF kappaB assay with sigma/deltamicro = 1.52. To better understand the relationship between cell-by-cell measurements and IC50 precision, 500 Monte Carlo simulations with varying cell-measurement SDs were used to explore three-, five-, seven-, and 11-point model titrations. The reductions in deltaIC50 90% confidence intervals from 11- to three-point titrations were 10-fold with the previously reported sigma/deltamicro = 1.52 and twofold with our sigma/deltamicro = 0.32. With these normalized parameters, this report provides a common statistical foundation, independent of the assay details, for evaluating the performance of imaging data on any instrument.